Improving fundamental abilities of atomic force microscopy for investigating quantitative nanoscale physical properties of complex biological systems
Abstract
Measurements of local material properties of complex biological systems (e.g. live cells and viruses) in their respective physiological conditions are extremely important in the fields of biophysics, nanotechnology, material science, and nanomedicine. Yet, little is known about the structure-function-property relationship of live cells and viruses. In the case of live cells, the measurements of progressive variations in viscoelastic properties in vitro can provide insight to the mechanistic processes underpinning morphogenesis, mechano-transduction, motility, metastasis, and many more fundamental cellular processes. In the case of living viruses, the relationship between capsid structural framework and the role of the DNA molecule interaction within viruses influencing their stiffness, damping and electrostatic properties can shed light in virological processes like protein subunits assembly/dissassembly, maturation, and infection. The study of mechanics of live cells and viruses has been limited in part due to the lack of technology capable of acquiring high-resolution (nanoscale, subcellular) images of its heterogeneous material properties which vary widely depending on origin and physical interaction. The capabilities of the atomic force microscope (AFM) for measuring forces and topography with sub-nm precision have greatly contributed to research related to biophysics and biomechanics during the past two decades. AFM based biomechanical studies have the unique advantage of resolving/mapping spatially the local material properties over living cells and viruses. However, conventional AFM techniques such as force-volume and quasi-static force-distance curves are too low resolution and low speed to resolve interesting biophysical processes such as cytoskeletal dynamics for cells or assembly/dissasembly of viruses. To overcome this bottleneck, a novel atomic force microscopy mode is developed, that leads to sub-10-nm resolution and sub-15-minutes mapping of local material properties of living cells and viruses in their respective physiological conditions. This advance is based on the harnessing of sub and superharmonic channels of cantilever vibration which are especially strong in liquids environments, which enable the mapping with exquisite detail of nanoscale material properties. Material properties such as storage and loss modulus or the spring and damping constant in live cells and the repulsive electrostatic force gradient, hydration layer viscosity and adhesion on viruses. By the use of this multi-harmonic dynamic AFM technique using a commercial AFM system, the local material properties of live rat fibroblast cells (RFB), red blood cells (RBC), human breast carcinoma cells (MDA-MB-231), and bacteriophage &phis;29 mature virions have been successfully imaged and extracted in relevant physiological conditions. Also, a novel high-speed dynamic AFM technique is developed to image at higher spatiotemporal resolution whole live cells under physiological conditions. This high-throughput technology enables the study of cellular processes in near real time frames, for example, the cytoskeleton structure dynamics of live fibroblast cells and human breast carcinoma cells. Overall, the contributions described in this thesis demonstrate the robustness and versatility of these novel advanced dynamic AFM techniques to investigate a wide range of complex biological relevant problems.
Degree
Ph.D.
Advisors
Raman, Purdue University.
Subject Area
Mechanics|Mechanical engineering|Nanoscience
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